4,7-dichlororhodamine dyes labeled polynucleotides

ABSTRACT

A set of 4,7-dichlororhodamine compounds useful as fluorescent dyes are disclosed having the structures 
                         
wherein R 1 -R 6  are hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, or, when taken together, R 1  and R 6  is benzo, or, when taken together, R 4  and R 5  is benzo; R 7 -R 10 , R 12 -R 16  and R 18  may be hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group; R 11  and R 17  may be hydrogen, lower alkyl, lower alkene, lower alkyne, phenyl, aryl, linking group; Y 1 -Y 4  are hydrogen, lower alkyl, or cycloalkyl, or, when taken together, Y 1  and R 2 , Y 2  and R 1  Y 3  and R 3 , and/or Y 4  and R 4  is propano, ethano, or substituted forms thereof; and X 1 -X 3  taken separately are hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonate, hydroxymethyl, and linking group, or any combinations thereof. In another aspect, the invention includes reagents labeled with the 4,7-dichlororhodamine dye compounds, including deoxynucleotides, dideoxynucleotides, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

FIELD OF THE INVENTION

This invention relates generally to fluorescent dye compounds useful asmolecular probes. More specifically, this invention relates to4,7-dichlororhodamine dyes useful as fluorescent labeling reagents.

REFERENCES

-   ABI PRISM™ 377 DNA Sequencer User's Manual, Rev. A, Chapter 2, The    Perkin-Elmer Corporation, Foster City, Calif. (p/n 903433) (1995).-   Bergot, J. B., etal., U.S. Pat. No. 5,366,860 (1994)-   Bergstrom, etal., JACS, 111: 374-375 (1989)-   Caskey etal., U.S. Pat. No. 5,364,759 (1994)-   Connell etal., Biotechniques, 5:342-348 (1987)-   Eckstein ed., Oligonucleotides and Analogs, Chapters 8 and 9, IRL    Press (1991)-   Eckstein, Oligonucleotides and Analogues, IRL Press (1991)-   Fung etal., U.S. Pat. No. 4,757,141 (1988)-   Fung etal., U.S. Pat. No. 4,855,225 (1989)-   Gait, Oligonucleotide Synthesis, IRL Press (1990)-   Gebeyehu etal, Nucleic Acids Research, 15:4513-4535 (1987)-   Gibson etal., Nucleic Acids Research, 15:6455-6467 (1987)-   Haralambidis etal, Nucleic Acids Research, 15:4856-4876 (1987)-   Haugland, Molecular Probes Handbook of Fluorescent Probes and    Research Chemicals, Molecular Probes, Inc. (1992)-   Herrmann, R., Josel, H., Drexhage, K. Arden, J., U.S. Pat. No.    5,750,409, issued May 12, 1998.-   Hermanson, Bioconjugate Techniques, Academic Press (1996)-   Hobbs etal., J. Org. Chem., 54:3420 (1989)-   Hobbs etal., U.S. Pat. No. 5,151,507 (1992)-   Hunkapiller, etal., U.S. Pat. No. 4,811,218 (1989)-   Innis etal. eds., PCR Protocols, Academic Press (1990)-   Ju etal., Proc. Natl. Acad. Sci. USA 92:4347-4351 (1995).-   Kasai, etal., Anal. Chem., 47:34037 (1975)-   Khan, S., Menchen, S., Rosenblum, B. “Substituted    propargylethoxyamido nucleosides, oligonucleotides and methods for    using same”, U.S. Pat. No. 5,770,716, issued Jun. 23, 1998-   Khan, S., Menchen, S., Rosenblum, B; “Propargylethoxyamino    nucleotides”, U.S. Pat. No. 5,821,356, issued Oct. 13, 1998-   Khan, S. etal, “Nucleotides including a rigid linker”, Ser. No.    09,172,789, filing date Oct. 14, 1998.-   Khanna, etal., U.S. Pat. No. 4,318,846 (1988)-   Lee etal. Nucleic Acids Research, 21:3761-3766 (1993)-   Lee, L., Benson, S., Rosenblum, B., Spurgeon, S., Cassel, J. and    Graham, R., “4,7-Dichlororhodamine Dyes”, U.S. Pat. No. 5,847,162,    issued Dec. 8, 1998-   Madabhushi, etal., International Patent Application No. WO    US94/13852 (1994)-   Maniatis, Methods in Enzymology, 65:299-305 (1980)-   Menchen, etal., U.S. Pat. No. 5,188,934 (1993)-   Mullis, U.S. Pat. No. 4,683,202 (1987)-   Nelson etal., Nucleosides and Nucleotides, 5(3):233-241 (1986)-   Nelson, et al., Nucleic Acids Research 20(23):6253-6259 (1992a)-   Nelson, U.S. Pat. No. 5,141,813 (1992b)-   Nelson, U.S. Pat. No. 5,401,837 (1995)-   Orgel etal., Nucleic Acids Research 11(18):6513 (1983)-   Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1    Springer-Verlag (1984)-   Pringle etal., DNA Core Facilities Newsletter, 1:15-21 (1988)-   Prober etal., Science, 238:336-341 (1987)-   Rickwood and Hames, eds., Gel Electrophoresis of Nucleic Acids: A    Practical Approach, IRL Press (1981)-   Sanger, etal., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)-   Scheit, Nucleotide Analogs, John Wiley (1980)-   Smith etal., Nucleic Acids Research, 113:2399-2412 (1985)-   Smith etal., U.S. Pat. No. 5,118,800 (1992)-   Steiner ed., Excited States of Biopolymers, Plenum Press (1983)-   Stryer, Biochemistry, W. H. Freeman (1981)-   Vos etal., Nucleic Acids Research, 23(21):4407-4414 (1995)-   Vogel, A, Rettig, W., Sens, R. Drexhage, K, Chemical Physics    Letters, 147:452-60 (1988)-   Ward, etal., U.S. Pat. No. 5,559,767 (1995)-   Webber, U.S. Pat. No. 5,075,217 (1991)-   Wheeless etal, Flow Cytometry: Instrumentation and Data Analysis,    pgs. 21-76, Academic Press (1985)-   Woo, etal., U.S. Pat. No. 5,231,191 (1993)

BACKGROUND

The non-radioactive detection of biological analytes is an importanttechnology in modern analytical biotechnology. By eliminating the needfor radioactive labels, safety is enhanced and the environmental impactof reagent disposal is greatly reduced, resulting in decreased costs foranalysis. Examples of methods utilizing such non-radioactive detectionmethods include DNA sequencing, oligonucleotide probe methods, detectionof polymerase-chain-reaction products, immunoassays, and the like.

In many applications the independent detection of multiple spatiallyoverlapping analytes in a mixture is required, e.g., single-tubemultiplex DNA probe assays, immuno assays, multicolor DNA sequencingmethods, and the like. In the case of multi-loci DNA probe assays, byproviding multicolor detection, the number of reaction tubes may bereduced thereby simplifying the experimental protocols and facilitatingthe manufacturing of application-specific kits. In the case of automatedDNA sequencing, multicolor labeling allows for the analysis of all fourbases in a single lane thereby increasing throughput over single-colormethods and eliminating uncertainties associated with inter-laneelectrophoretic mobility variations.

Multiplex detection imposes a number of severe constraints on theselection of dye labels, particularly for analyses requiring anelectrophoretic separation and treatment with enzymes, e.g., DNAsequencing. First, it is difficult to find a collection of dyes whoseemission spectra are spectrally resolved, since the typical emissionband half-width for organic fluorescent dyes is about 40-80 nanometers(nm) and the width of the available spectrum is limited by theexcitation light source. Second, even if dyes with non-overlappingemission spectra are found, the set may still not be suitable if therespective fluorescent efficiencies are too low. For example, in thecase of DNA sequencing, increased sample loading cannot compensate forlow fluorescence efficiencies (Pringle). Third, when several fluorescentdyes are used concurrently, simultaneous excitation becomes difficultbecause the absorption bands of the dyes are widely separated. Fourth,the charge, molecular size, and conformation of the dyes must notadversely affect the electrophoretic mobilities of the fragments.Finally, the fluorescent dyes must be compatible with the chemistry usedto create or manipulate the fragments, e.g., DNA synthesis solvents andreagents, buffers, polymerase enzymes, ligase enzymes, and the like.

Because of these severe constraints only a few sets of fluorescent dyeshave been found that can be used in multicolor applications,particularly in the area of four-color DNA sequencing (Smith 1992, 1995;Prober, Connell).

One class of fluorescent dyes particularly useful in multicolorapplications are the rhodamine dyes, e.g., tetramethylrhodamine (TAMRA),rhodamine X (ROX), rhodamine 6G (R6G), rhodamine 110 (R110), and thelike (Bergot). Rhodamine dyes are particularly attractive relative tofluorescein dyes because (1) rhodamines are typically more photostablethan fluoresceins, (2) rhodamine-labeled dideoxynucleotides are bettersubstrates for thermostable polymerase enzymes, and (3) the emissionspectra of rhodamine dyes is significantly to the red (higherwavelength) of fluoresceins.

However, one important drawback of presently available rhodamine dyes inthe context of multiplex detection methods is the relatively broademission spectrum of such dyes. This broad emission spectrum results inpoor spectral resolution between spectrally neighboring dyes therebymaking the multicomponent analysis of such dye combinations difficult.The fluorescence emission spectra shown in FIG. 7A demonstrate this highdegree of spectral overlap. A second drawback of currently availablerhodamine dyes is that their absorption spectrum does not match thewavelength of currently available solid state frequency-doubled greendiode lasers, e.g., neodymium solid-state YAG lasers, which have anemission line at approximately 532 mm. It is highly advantageous to usesuch lasers because of their compact size, long useful life, andefficient use of power.

SUMMARY

The present invention is directed towards our discovery of a class of4,7-dichlororhodamine dyes useful as molecular probes.

It is an object of the invention to provide a class of rhodamine dyeswhich have emission spectra which are substantially narrower thanpresently available rhodamine dyes.

It is another object of the invention to provide a class of rhodaminedyes which have an absorption spectrum shifted to the red as compared toexisting rhodamine dyes.

In a first aspect, the foregoing and other objects of the invention areachieved by a compound having the formula:

In a second aspect, the invention includes a compound having theformula:

In a third aspect the invention includes a labeled nucleotide having theformula:

In a fourth aspect the invention includes a labeled polynucleotidecontaining a nucleotide having the formula:

The linkage linking B, and D is attached to D at one of positions R₁-R₆or X₁-X₃. Preferably, the linkage linking B and D is attached to D atone of positions X₂ or X₃. In a particularly preferred embodiment, thelinkage is

If B is a purine, the linkage is attached to the 8-position of thepurine. If B is 7-deazapurine, the linkage is attached to the 7-positionof the 7-deazapurine. If B is pyrimidine, the linkage is attached to the5-position of the pyrimidine.

In a fifth aspect, the present invention includes a method ofpolynucleotide sequencing, such method including the following steps.Forming a mixture of a first, a second, a third, and a fourth class ofpolynucleotides such that each polynucleotide in the first classincludes a 3′-terminal dideoxyadenosine and is labeled with a first dye,each polynucleotide in the second class includes a 3′-terminaldideoxycytidine and is labeled with a second dye, each polynucleotide inthe third class includes a 3′-terminal dideoxyguanosine and is labeledwith a third dye, and, each polynucleotide in the fourth class includesa 3′-terminal dideoxythymidine and is labeled with a fourth dye. Thedyes are selected such that one of the first second, third, or fourthdyes is a 4,7-dichlororhodamine dye of the invention, the other of thedyes being spectrally resolvable from the 4,7-dichlororhodamine dye andfrom each other. Electrophoretically separating the polynucleotidesthereby forming bands of similarly sized polynucleotides, illuminatingthe bands with an illumination beam capable of causing the dyes tofluoresce, and, identifying th classes of the polynucleotides in thebands by the fluorescence spectrum of the dyes.

These and other aspects, objects, features, and advantages of thepresent invention will become better understood with reference to thefollowing description, drawings, and appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

Generally, the present invention comprises a novel class of4,7-dichlororhodamine compounds useful as fluorescent dyes, reagentsemploying such dyes as molecular labels, and methods utilizing such dyesand reagents in the area of analytical biotechnology. The compounds ofthe present invention find particular application in the area ofmulticolor fluorescent DNA sequencing and fragment analysis.

The invention is based in part on the discovery that the fluorescentproperties of 4,7-dichlororhodamines and related dyes are highlyfavorable for use as molecular probes. Their emission band widths aregenerally 20-30 percent narrower than analogs lacking the 4,7-dichloroderivatives, and, their emission and absorption maxima are atwavelengths generally about 10-30 nm higher than analogs lacking the4,7-dichloro derivatives.

I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Linking group” (L) refers to a functionality capable of reacting with a“complementary functionality” attached to a reagent, such reactionforming a “linkage” connecting a dye to a reagent. The particularlinking group used depends on the nature of the complementaryfunctionality and the type of linkage desired. In some cases, thelinking group must be activated prior to reaction with a complementaryfunctionality, e.g., the activation of a carboxylate linking group withdicyclohexylcarbodiimide and N-hydroxysuccinimide to form aN-hydroxysuccinimide (NHS) ester. Preferably, whenever the complementaryfunctionality is amine, the linking group of the invention isisothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride,aldehyde or glyoxal, epoxide, carbonate, aryl halide, imidoester,carbodiimide, anhydride, 4,6-dichlorotriazinylamine, or other activecarboxylate. Preferably, whenever the complementary functionality issulfhydryl, the linking group is haloacetyl, alkyl halide, maleimide,halo acetyl, aziridine, acryloyl, arylating agent, e.g., fluorobenzene,and the like. When the complementary functionality is carboxylate, thelinking group is preferably diazoalane, diazoacetyl,carbonyldiimidazole, and carbodiimide (Hermanson). In a particularlypreferred embodiment, the linking group is an activated NHS ester whichreacts with an amine complementary functionality, where to form theactivated NHS ester, a dye of the invention including a carboxylatelinking group is reacted with dicyclohexylcarbodiimide andN-hydroxysuccinimide to form the NHS ester (Khanna; Kasai). Table 1below shows a sampling of representative linking groups along withcompatible complementary functionalities and resulting linkages.

TABLE 1 Comple- mentary Linking Group Functionality Linkage —NCS —NH₂—NHCSNH—

—NH₂

—SO₂X —NH₂ —SO₂NH—

—NH₂

—SH

—SH

The term “lower alkyl” denotes straight-chain and branched hydrocarbonmoieties containing from 1 to 8 carbon atoms, i.e., methyl, ethyl,propyl, isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl,tert-pentyl, and the like.

The term “propano” in particular refers to the moiety —CH₂CH₂CH₂—.

“Cycloalkyl” refers to hydrocarbon moieties that form rings, e.g.cyclohexyl and cyclopentyl. Nitrogen atoms with cycloalkyl substituentsmay form aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, largerrings, and substituted forms thereof.

“Lower substituted alkyl” denotes a lower alkyl includingelectron-withdrawing substituents, such as halo, cyano, nitro, sulfo,and the like.

“Lower haloalkyl” denotes a lower substituted alkyl with one or morehalogen atom substituents, usually fluoro, chloro, bromo, or iodo.

“Lower alkene” denotes a lower alkyl or lower substituted alkyl whereinone or more of the carbon-carbon bonds is a double bond.

“Lower alkyne” denotes a lower alkyl or lower substituted alkyl whereinone or more of the carbon-carbon bonds is a triple bond.

“Lower Alkoxy” refers to a moiety including lower alkyl single bonded toan oxygen atom.

“Aryl” refers to single or multiple phenyl or substituted phenyl, e.g.,benzene, naphthalene, anthracene, biphenyl, and the like.

The term “nucleoside” refers to a compound consisting of a purine,deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine,cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like,linked to a pentose at the 1′position, including 2′-deoxy and2′-hydroxyl forms (Stryer). The term “nucleotide” as used herein refersto a phosphate ester of a nucleoside, e.g., triphosphate esters, whereinthe most common site of esterification is the hydroxyl group attached atthe C-5 position of the pentose. Many times in the present disclosurethe term nucleoside will be intended to include both nucleosides andnucleotides.

“Analogs” in reference to nucleosides include synthetic analogs havingmodified base moieties, modified sugar moieties, and/or modifiedphosphate ester moieties, e.g., as described elsewhere (Scheit;Eckstein). The term “labeled nucleoside” refers to nucleosides which arecovalently attached to the dye compounds of Formula I.

As used herein, the terms “polynucleotide” or “oligonucleotide” refer tolinear polymers of natural nucleotide monomers or analogs thereofincluding double and single stranded deoxyribonucleotides,ribonucleotides, α-anomeric forms thereof, and the like. Usually thenucleoside monomers are linked by phosphodiester linkages, where as usedherein, the term “phosphodiester linkage” refers to phosphodiester bondsor analogs thereof including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H⁺, NH₄ ⁺, Na⁺, and the like if such counterions arepresent. Polynucleotides typically range in size from a few monomericunits, e.g. 8-40, to several thousands of monomeric units. Whenever apolynucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine,unless otherwise noted.

As used herein the term “spectral resolution” in reference to a set ofdyes means that the fluorescent emission spectra of the dyes aresufficiently distinct, i.e., sufficiently non-overlapping, that reagentsto which the respective dyes are attached, e.g., polynucleotides, can bedistinguished on the basis of the fluorescent signal generated by therespective dyes using standard photodetection systems, e.g., employing asystem of band pass filters and photomultiplier tubes, a charged-coupleddevice in conjunction with a spectrograph, or the like, as exemplifiedby systems described elsewhere (Hunkapiller, Wheeless).

The term “substituted” as used herein refers to a molecule wherein oneor more hydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example, an unsubstituted nitrogen is—NH₂, while a substituted nitrogen is —NHCH₃. Exemplary substituentsinclude but are not limited to halo, e.g., fluorine and chlorine, loweralkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, amino,ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl,polycyclic aromatic, heterocycle, and linking group.

II. 4,7-Dichlororhodamine Dye Compounds.

In a first aspect, the present invention comprises a novel class of4,7-dichlororhodamine dye compounds having the general structure shownimmediately below as Formula I. (Note that all molecular structuresprovided throughout this disclosure are intended to encompass not onlythe exact electronic structure presented, but also include all resonantstructures, enantiomers, diastereomers, and protonation states thereof.)

In Formula I, the variable substituents are defined as follows. R₁-R₆taken separately are hydrogen, fluorine, chlorine, lower alkyl, loweralkene, lower alkyne, cycloalkyl, phenyl, aryl, sulfonate, sulfone,amino, amido, nitrile, lower alkoxy, linking group, or combinationsthereof, or, when taken together, R₁ and R₆ is benzo, or, when takentogether, R₄ and R₅ is benzo. Preferably, R₁-R₆ are hydrogen, methyl, orethyl. Y₁-Y₄ taken separately are hydrogen, lower alkyl, alkylsulfonate, alkyl carboxylate, or cycloalkyl. Or, when taken together, Y₁and R₂, Y₂ and R₁, Y₃ and R₃, and/or Y₄ and R₄ are propano, ethano, orsubstituted forms thereof to form fused rings. X₁-X₃ taken separatelyare hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonicacid (sulfonate), hydroxymethyl (—CH₂OH), and linking groups.Preferably, X₁ is carboxylate and X₂ and X₃ taken separately arehydrogen and linking group.

In a set of particularly preferred compound of the present invention, Ris hydrogen (dJON) or methyl (DMDJ), shown below as FORMULA II.

Another particularly preferred compound of the present invention isreferred to as dR650, shown below as FORMULA III.

A third particularly preferred set of compounds are where R is6-hexanoic acid (dJODA) or methyl-p-benzoic acid (dR134), as shown belowin FORMULA IV.

A fourth particularly preferred compound of the present invention isreferred to herein as dR139, where Y₁ and Y₂, and Y₃ and Y₄, formpyrrolidinyl rings as nitrogen substituents, R₁₋₆ are hydrogen, X₁ iscarboxyl, and X₂ and X₃ are carboxyl and hydrogen. The structure ofdR139 is shown below as Formula V.

In a second aspect, the present invention comprises a novel class of4,7-dichlororhodamie dye compounds having the general structure shownbelow as FORMULA VI.

In Formula VI, R₇-R₁₀, R₁₂-₁₆, and R₁₈ may be hydrogen, fluorine,chlorine, methyl, ethyl, lower alkyl, lower alkene, lower alkyne,cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino, amido, nitrile,lower alkoxy, linking group, or combinations thereof. Preferably, R₇-R₁₀and R₁₃-R₁₆ are hydrogen, methyl, or ethyl. R₇ and R₈, or R₁₃ and R₁₄,taken together may be oxygen (═O), sulfur (═S), imminium (═NH),alkylimminium (═NR). R₁₁ and R₁₇ may be hydrogen, lower alkyl, alkylsulfonate, alkyl carboxylate, lower alkene, lower alkyne, cycloalkyl,phenyl, aryl, linking group, or combinations thereof. Preferably R₁₁ andR₁₇ are methyl or phenyl. X₁-X₃ taken separately are hydrogen, chlorine,fluorine, lower alkyl, amine, amide, carboxylate, sulfonic acid(sulfonate), hydroxymethyl (—CH₂OH), and linking groups. Preferably, X₁is carboxylate and X₂ and X₃ taken separately are hydrogen or linkinggroup. Particular preferred embodiments are where R₇, R₈, R₁₀ R₁₃, R₁₄,and R₁₇ are hydrogen or methyl, R₉ and R₁₅ are hydrogen, R₁₁ and R₁₅ aremethyl or phenyl, and R₁₂ and R₁₈ are hydrogen.

III. Reagents Utilizing 4,7-Dichlororhodamine Dye Compounds

In another aspect, the present invention comprises reagents labeled withthe 4,7-dichlororhodamine dye compounds of Formulas I-VI. Reagents ofthe invention can be virtually anything to which the dyes of theinvention can be attached. Preferably the dyes are covalently attachedto the reagent directly or through a linkage. Exemplary reagents includeproteins, polypeptides, polysaccharides, nucleotides, nucleosides,polynucleotides, lipids, solid supports, organic and inorganic polymers,and combinations and assemblages thereof, such as chromosomes, nuclei,living cells, such as bacteria, other microorganisms, mammalian cells,tissues, glycoproteins, and the like.

A. Nucleotide Reagents

A preferred class of reagents of the present invention comprisenucleotides and nucleosides which incorporate the asymmetricbenzoxanthene dyes of the invention. Such nucleotide/side reagents areparticularly useful in the context of labeling polynucleotides formed byenzymatic synthesis, e.g., nucleotide triphosphates used in the contextof PCR amplification, Sanger-type polynucleotide sequencing, andnick-translation reactions.

Preferred nucleotide/side reagents of the present invention are shownbelow in Formula VII wherein

B is a nucleoside base; 7-deazapurine, purine, or pyrimidine nucleotidebase, analogs thereof, and preferably uracil, cytosine, deazaadenine, ordeazaguanosine. D is the 4,7-dichlororhodamine dye compound of theFormulas I-VI of the invention. W₁ and W₂ taken separately are H or OH.W₃ is OH, OPO₃, OP₂O₆, OP₃O₉, including analogs thereof, e.g.,phosphorothioate, phosphoroanilidate, phosphoroanilothioate,phosphoramidiate, and other like phosphate analogs, including associatedcounterions if present, e.g., H⁺, Na⁺, NH₄ ⁺, and the like. In onepreferred embodiment, W₁ is H, W₂ is OH, and W₃ is OP₃O₉. In a secondpreferred embodiment, W₁ and W₂ are H and W₃ is OP₃O₉. When B is purineor 7-deazapurine, the sugar moiety is attached at the N⁹-position of thepurine or deazapurine, and when B is pyrimidine, the sugar moiety isattached at the N¹-position of the pyrimidine. The linkage linking B andD is attached to D at one of positions R₁-R₁₈ or X₁-X₃.

When B is purine or 7-deazapurine, the sugar moiety is attached at theN⁹-position of the purine or deazapurine, and when B is pyrimidine, thesugar moiety is attached at the N¹-position of the pyrimidine.

The linkage linking B and D may be attached to D at any one of positionsR₁-R₁₈ or X₁-X₃. Preferably, the linkage is attached at one of X₂ or X₃.Preferably, when B is a purine, the linkage linking B and D is attachedto the 8-position of the purine, when B is 7-deazapurine, the linkage isattached to the 7-position of the 7-deazapurine, and when B ispyrimidine, the linkage is attached to the 5-position of the pyrimidine.

In one particularly preferred embodiment, the nucleotides of the presentinvention are dideoxynucleotide triphosphates having the structure shownbelow in Formula VIII, including associated counterions if present.

Labeled dideoxy nucleotides such as that shown in Formula VIII findparticular application as chain terminating agents, or “terminators”, inSanger-type DNA sequencing methods (Sanger).

In a second particularly preferred embodiment, the nucleotides of thepresent invention are deoxynucleotide triphosphates having the structureshown in Formula IX below, including associated counterions if present.

Labeled deoxynucleotides such as that shown in Formula IX findparticular application as means for labeling polymerase extensionproducts, e.g., in the polymerase chain reaction (Mullis).

Nucleotide/side labeling can be accomplished using any of a large numberof known nucleoside/tide labeling techniques using known linking groups,and associated complementary functionalities to form linkages. See abovefor a discussion of preferred linking groups. The linkage linking thedye and nucleoside should (i) not interfere with oligonucleotide-targethybridization, (ii) be compatible with relevant enzymes, e.g.,polymerases, ligases, and the like, and (iii) not quench thefluorescence of the dye.

In one preferred embodiment, the dyes of the invention are covalentlylinked to the 5-carbon of pyrimidine bases or to the 7-carbon of7-deazapurine bases. Several suitable base labeling procedures have beenreported that can be used with the invention. (Gibson; Gebeyehu;Haralambidis; Nelson 1992; Bergstrom; Fung 1988; Ward; Woo.)

Preferably, the linkages are acetylenic amido or alkenic amido linkages,the linkage between the dye and the nucleotide base being formed byreacting an activated NHS ester of the dye with an alkynylamino- oralkenylamino-derivatized base of a nucleotide. More preferably, theresulting linkage is 3-carboxy)amino-1-propynyl or 3-amino-1-propyn-1-yl(Formula X.1). Several preferred linkages for linking the dyes of theinvention to a nucleoside base are shown below as Formulas X.1-6 (Khan).

The synthesis of alkynylamino-derivatized nucleosides is described by(Hobbs 1989, 1992). Briefly, the alkynylamino-derivatized nucleotidesare formed by placing the appropriate halodideoxynucleoside (usually5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides) and Cu(I)in a flask, flushing with argon to remove air, adding dry DMF, followedby addition of an alkynylamine, triethylamine and Pd°. The reactionmixture is stirred for several hours, or until thin layer chromatographyindicates consumption of the halodideoxynucleoside. When an unprotectedalkynylamine is used, the alkynylamino-nucleoside can be isolated byconcentrating the reaction mixture and chromatographing on silica gelusing an eluting solvent which contains ammonium hydroxide to neutralizethe hydrohalide generated in the coupling reaction. When a protectedalkylamine is used, methanol/methylene chloride can be added to thereaction mixture, followed by the bicarbonate form of a strongly basicanion exchange resin. The slurry can then be stirred for about 45minutes, filtered, and the resin rinsed with additionalmethanol/methylene chloride. The combined filtrates can be concentratedand purified by flash-chromatography on silica gel using amethanol-methylene chloride gradient. The 5′-triphosphates are obtainedby standard techniques.

B. Polynucleotide Reagents

Yet another preferred class of reagents of the present inventioncomprise polynucleotides labeled with the 4,7-dichlororhodamine dyes ofthe invention. Such labeled polynucleotides are useful in a number ofimportant contexts including as DNA sequencing primers, PCR primers,oligonucleotide hybridization probes, and the like.

The polynucleotides of the invention include a nucleotide having theformula:

wherein the variable substituents and linkages are defined as follows. Dis a 4,7-dichlororhodamine dye compound of the invention. B is a7-deazapurine, purine, or pyrimidine nucleotide base, preferably uracil,cytosine, deazaadenine, or deazaguanosine. Z₁ is H, OH, or OCH₃. Z₂ isH, OH, OPO₃, OP₂O₆, OP₃O₉, or Nuc, a neighboring nucleotide, wherein Nucand the nucleoside are linked by a phosphodiester linkage or analogthereof, e.g., phosphorothioate, phosphoroanilidate,phosphoroanilothioate, phosphoramidiate, and other like phosphateanalogs, including associated counterions if present, e.g., H⁺, Na⁺, NH₄⁺, the linkage being attached to the 5′-position of Nuc. Z₃ is H, OPO₂,including phosphate analogs, or Nuc, wherein Nuc and the nucleoside arelinked by a phosphodiester linkage or analog thereof, the linkage beingattached to the 3′-position of Nuc wherein Nuc refers to a nucleoside,nucleotide, or polynucleotide. When B is purine or 7-deazapurine, thesugar moiety is attached at the N⁹-position of the purine ordeazapurine, and when B is pyrimidine, the sugar moiety is attached atthe N¹-position of the pyrimidine. B is attached to the sugar moiety andto the dye compound as described above for the nucleotide reagent of theinvention. As defined, the labeled nucleotide of Formula XI can be the5′-terminal nucleotide, the 3′-terminal nucleotide, or any internalnucleotide of the polynucleotide.

In one preferred embodiment, the polynucleotide of the present inventionincludes multiple dyes, at least one of which is a dye compound of theinvention, located such that fluorescence energy transfer takes placebetween a donor dye and an acceptor dye. Such multi-dye polynucleotidesfind application as spectrally-tunable probes or DNA sequencing primers(Ju; Lee).

Labeled polynucleotides may be synthesized either enzymatically, e.g.,using a DNA polymerase or ligase (Stryer), or by chemical synthesis,e.g., by the phosphoramidite method, the phosphite-triester method, andthe like (Gait). Labels may be introduced during enzymatic synthesisutilizing labeled nucleotide triphosphate monomers as described above ormay be introduced subsequent to synthesis.

Generally, if the labeled polynucleotide is made by enzymatic synthesis,the following procedure may be used. A template DNA is denatured and anoligonucleotide primer is annealed to the template DNA. A mixture ofdeoxynucleotide triphosphates and/or dideoxynucleotide triphosphates isadded to the reaction including dGTP, dATP, dCTP, ddTTP, ddGTP, ddATP,ddCTP, and ddTTP, where at least a fraction of one of at least one thedeoxynucleotides and/or dideoxynucleotides is labeled with a dyecompound of the invention as described above. Next, a polymerase enzymeis added under-conditions where its polymerase activity is operative. Alabeled polynucleotide is formed by the incorporation of the labeleddeoxynucleotides and/or dideoxynucleotides during polymerase strandsynthesis. In an alternative enzymatic synthesis method, two primers areused instead of one, one primer complementary to the + (plus) strand andthe other complementary to the − (minus) strand of the target, thepolymerase is a thermostable polymerase, and the reaction temperature iscycled between a denaturation temperature and an extension temperature,thereby exponentially synthesizing a labeled complement to the targetsequence by PCR (Mullis; Innis).

Subsequent to synthesis, the polynucleotide may be labeled at a numberof positions including the 5′-terminus (Eckstein; Orgel; Smith); thephosphodiester backbone (Eckstein); or at the 3′-terminus (Nelson 1992a;Nelson 1992b; Nelson 1995). For a through review of oligonucleotidelabeling procedures see (Steiner).

In one preferred post-synthesis chemical labeling method anoligonucleotide is labeled as follows. A dye including a carboxylatelinking group is converted to the NHS ester by reacting withapproximately 1 equivalent of 1,3-dicyclohexylcarbodiimide andapproximately 3 equivalents of N-hydroxysuccinimide in dry ethyl acetatefor 3 hours at room temperature. The reaction mixture is washed with 5%HCl, dried over magnesium sulfate, filtered, and concentrated to a solidwhich is resuspended in DMSO. The DMSO dye stock is then added in excess(10-20×) to an aminohexyl derivatized oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours(Fung 1988). The dye labeled oligonucleotide is separated from unreacteddye by passage through a size-exclusion chromatography column elutingwith buffer, e.g., 0.1 molar triethylammonium acetate (TEAA). Thefraction containing the crude labeled oligonucleotide is furtherpurified by reverse phase HPLC employing gradient elution.

IV. Methods Utilizing Compounds and Reagents of the Invention

The dyes and reagents of the present invention are well suited tomethods utilizing fluorescent detection, particularly methods requiringthe simultaneous detection of multiple spatially-overlapping analytes.Dyes and reagents of the invention are particularly well suited foridentifying classes of polynucleotides that have been subjected to abiochemical separation procedure, such as electrophoresis, where aseries of bands or spots of target substances having similarphysiochemical properties, e.g. size, conformation, charge,hydrophobicity, or the like, are present in a linear or planararrangement. As used herein, the term “bands” includes any spatialgrouping or aggregation of analytes on the basis of similar or identicalphysiochemical properties. Usually bands arise in the separation ofdye-polynucleotide conjugates by electrophoresis.

Classes of polynucleotides can arise in a variety of contexts. In apreferred category of methods referred to herein as “fragment analysis”or “genetic analysis” methods, labeled polynucleotide fragments aregenerated through template-directed enzymatic synthesis using labeledprimers or nucleotides, e.g., by ligation or polymerase-directed primerextension; the fragments are subjected to a size-dependent separationprocess, e.g., electrophoresis or chromatography; and, the separatedfragments are detected subsequent to the separation, e.g., bylaser-induced fluorescence. In a particularly preferred embodiment,multiple classes of polynucleotides are separated simultaneously and thedifferent classes are distinguished by spectrally resolvable labels.

One such fragment analysis method known as amplified fragment lengthpolymorphism detection (AmpFLP) is based on amplified fragment lengthpolymorphisms, i.e., restriction fragment length polymorphisms that areamplified by PCR (Vos). These amplified fragments of varying size serveas linked markers for following mutant genes through families. Thecloser the amplified fragment is to the mutant gene on the chromosome,the higher the linkage correlation. Because genes for many geneticdisorders have not been identified, these linkage markers serve to helpevaluate disease risk or paternity. In the AmpFLPs technique, thepolynucleotides may be labeled by using a labeled polynucleotide PCRprimer, or by utilizing labeled nucleotide triphosphates in the PCR.

Another exemplary fragment analysis method is based on variable numberof tandem repeats; or VNTRs (Webber; Caskey). VNTRs are regions ofdouble-stranded DNA that contain adjacent multiple copies of aparticular sequence, with the number of repeating units being variable.Examples of VNTR loci are pYNZ22, pMCT118, and Apo B. A subset of VNTRmethods are those methods based on the detection of microsatelliterepeats, or short tandem repeats (STRs), i.e., tandem repeats of DNAcharacterized by a short (2-4 bases) repeated sequence. One of the mostabundant interspersed repetitive DNA families in humans is the(dC-dA)n-(dG-dT)n dinucleotide repeat family (also called the (CA)ndinucleotide repeat family). There are thought to be as many as 50,000to 100,000 (CA)n repeat regions in the human genome, typically with15-30 repeats per block. Many of these repeat regions are polymorphic inlength and can therefore serve as useful genetic markers. Preferably, inVNTR or STR methods, a dye label is introduced into the polynucleotidefragments by using a dye-labeled PCR primer.

In a particularly preferred fragment analysis method, classes identifiedin accordance with the invention are defined in terms of terminalnucleotides so that a correspondence is established between the fourpossible terminal bases and the members of a set of spectrallyresolvable dyes (Fung 1989). Such sets are readily assembled from thedyes of the invention by measuring emission and absorption bandwidthswith commercially available spectrophotometers. More preferably, theclasses arise in the context of the chemical or chain terminationmethods of DNA sequencing, and most preferably the classes arise in thecontext of the chain termination method, i.e., dideoxy DNA sequencing,or Sanger sequencing. This method involves the synthesis of a DNA strandby a DNA polymerase in vitro using a single-stranded or double-strandedDNA template whose sequence is to be determined. Synthesis is initiatedat only the one site where an oligonucleotide primer anneals to thetemplate. The synthesis reaction is terminated by incorporation of anucleotide analog that will not support continued DNA elongation. Thechain-terminating nucleotide analogs are typically2′,3′-dideoxynucleoside 5′-triphosphates (ddNTPs) which lack the 3′-OHgroup necessary for 3′ to 5′ DNA chain elongation. When properproportions of dNPTs (2′-deoxynucleoside 5′-triphosphates) and one ofthe four ddNTPs are used, enzyme-catalyzed polymerization will beterminated in a fraction of the population of chains at each site wherethe ddNTP can be incorporated. If labeled primers or labeled ddNTPs areused for each reaction, the sequence information can be detected byfluorescence after separation by high-resolution electrophoresis. In thechain termination method, dyes of the invention can be attached toeither sequencing primers or dideoxynucleotides.

In each of the above fragment analysis methods labeled polynucleotidesare preferably separated by electrophoretic procedures (Rickwood andHames; Osterman) Preferably the type of electrophoretic matrix iscrosslinked or uncrosslinked polyacrylamide having a concentration(weight to volume) of between about 2-20 weight percent. Morepreferably, the polyacrylamide concentration is between about 4-8percent. Preferably in the context of DNA sequencing in particular, theelectrophoresis matrix includes a strand separating, or denaturing,agent, e.g., urea, formamide, and the like. Detailed procedures forconstructing such matrices are given by (Maniatis 1980; ABI PRISM™ 377DNA Sequencer User's Manual). The optimal polymer concentration, pH,temperature, concentration of denaturing agent, etc. employed in aparticular separation depends on many factors, including the size rangeof the nucleic acids to be separated, their base compositions, whetherthey are single stranded or double stranded, and the nature of theclasses for which information is sought by electrophoresis. Accordinglyapplication of the invention may require standard preliminary testing tooptimize conditions for particular separations.

Subsequent to electrophoretic separation, the dye-polynucleotideconjugates are detected by measuring the fluorescence emission from thedye labeled polynucleotides. To perform such detection, the labeledpolynucleotides are illuminated by standard means, e.g. high intensitymercury vapor lamps, lasers, or the like. Preferably the illuminationmeans is a laser having an illumination beam at a wavelength between 488and 550 nm. More preferably, the dye-polynucleotides are illuminated bylaser light generated by an argon ion laser, particularly the 488 and514 nm emission lines of an argon ion laser, or the 532 emission line ofa neodymium solid-state YAG laser. Several argon ion lasers areavailable commercially which lase simultaneously at these lines, e.g.,the Model 2001 from Cyonics, Ltd. (Sunnyvale, Calif.). The fluorescenceis then detected by a light-sensitive detector, e.g., a photomultipliertube, a charged coupled device, or the like.

V. EXAMPLES

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope. All reagents werepurchased from Aldrich Chemical Co. (Milwaukee, Wis.) except asotherwise indicated. The 3,6-dichlorotrimellitic anhydride was preparedas described by (Khanna).

Example 1

Preparation of dR139:

A solution of m-aminophenol (12.6 gm, 0.115 moles) and 1,4-dibromobutane(50 gm, 0.23 moles) was heated to 130° C. for 12 hr. The mixture wascooled to room temperature and triturated with diethylether and thenethyl acetate. The residue was dissolved in ethyl acetate and extractedwith 1M NaOH, water, and sat. NaCl. After drying the organic layer withMgSO₄, filtering, and evaporating solvent under vacuum, the crudeproduct was purified by silica gel chromatography to give a pale yellowsolid. The solid was refluxed with 500 ml toluene and 17 mltriethylamine (0.12 moles) for one hr, cooled to room temperature andwashed with water and sat. NaCl. The solution was dried again overMgSO₄, filtered, and evaporated under vacuum to give3-pyrrolidinylphenol as a white solid (6.0 gm, 0.037 moles, 32%).

A mixture of 3-pyrrolidinylphenol (1.63 gm, 10 mmol),3,6-dichlorotrimellitic anhydride (1.3 g, 5 mmol) and polyphosphoricacid (5 mL) was heated to 160° C. for 2 hr. After cooling to roomtemperature, water (20 ml) was added and the precipitated product wascollected by filtration. Purification by reverse-phase HPLC separatedthe 5 and 6 carboxyl isomers of dR139, Abs. max 568 nm (methanol),(Formula V), Isomer 1 (0.3 gm, 5%) and Isomer 2 (0.6 gm, 10%).

Example 2

Synthesis of dR650

4-Hydroxyindole was reduced with sodium cyanoborohydride and acetic acidto give 4-hydroxy, dihydroindole. A mixture of ethyl iodide (40 ml),potassium carbonate (1.09 gm, 7.8 mmole) and 4-hydroxy, dihydroindole(1.06 gm, 7.8 mmole) was refluxed for 2 hr. Excess ethyl iodide wasevaporated under vacuum, water was added (10 ml) and the product wasextracted with dichloromethane. Silica gel chromatography gaveN-ethyl-4-hydroxy-dihydroindole (0.30 gm, 23% yield) as a pale yellowsolid.

A mixture of N-ethyl-4-hydroxy-dihydroindole (0.30 g, 1.8 mmol),3,6-dichlorotrimellitic anhydride (230 mg, 0.9 mmol) and polyphosphoricanhydride (PPA) (5 g) was heated to 180° C. for 2.5 hr. After cooling toroom temperature, the solid was dissolved in aqueous NaOH (1M, 7.5 ml).The product was precipitated with aqueous HCl (2M, 7.5 ml). The solidwas collected by filtration and purified by reverse-phase HPLC to givedR650, Em. max, 633 nm (8M urea), Abs. max. 614 nm (8M urea), (FORMULAIII). The regiochemistry of the 5 and 6 carboxyl groups of the isomerswere not assigned. Isomer 1 (53 mg, 5%) and Isomer 2 (105 mg, 10%) wereseparable by reverse-phase HPLC.

Example 3

Synthesis of dJON

A mixture of 6-hydroxy-1,2,3,4-tetrahydroquinoline (1.49 gm, 10-mmol),3,6-dichlorotrimellitic anhydride (1.3 g, 5 mmol) and methanesulfonicacid (2 ml) was heated to 160° C. for 6 hr. After cooling to roomtemperature, water (20 ml) was added and the precipitated product wascollected by filtration. Purification by reverse-phase HPLC separatedthe 5 and 6 carboxyl isomers of dJON, Abs. max 557 nm (methanol),(Formula II), Isomer 1 (270 mg, 5%) and Isomer 2 (550 mg, 10%).

Example 4

Synthesis of Formula VI

A solution of O-fluoro-benzoyl chloride (1.59 gm, 10 mmole) in 2 mldichloromethane was added dropwise to m-N-methylamino phenol (1.23 gm,10 mmole) in 5 ml dichloromethane and triethylamine (1.01 g, 1 mmole)cooled in an ice bath. The reaction was allowed to warm to roomtemperature over one hour. The mixture was diluted with dichloromethane,extracted with water and sat. NaCl, dried with MgSO₄, filtered, andevaporated under vacuum. The crude product was purified by silica gelchromatography to give N-methyl, N-(m-hydroxyphenyl)-O-fluoro-benzamideas a white foam (1.2 gm, 5 mmole, 50%).

Sodium hydride (200 mg, 60% dispersion in oil, 5 mmol) was added to theamide (1.2 gm, 5 mmole) in 10 ml dimethylformamide at ambienttemperature. The mixture was then refluxed for 2 hours and cooled toroom temperature. Solvent was evaporated under vacuum and 5 mlhydrochloric acid (2M) was added. The mixture was extracted with ethylacetate twice. The combined ethyl acetate extracts were washed withwater and sat. NaCl, dried with MgSO4, filtered, and evaporated undervacuum. The crude product was purified by silica gel chromatography togive the cyclized, tricyclic amide as a white solid (0.56 gm, 2.5 mmole,50%).

Dimethyl sulfide/borane complex (3.75 ml, 7.5 mmole, 2M in THF) wasadded dropwise to the tricyclic amide (0.56 gm, 2.5 mmole) in 10 ml drytetrahydrofuran cooled in an ice bath. The mixture was refluxed for onehour, cooled in an ice bath, and 10 ml methanol was added slowly.Solvent was evaporated under vacuum, more methanol was added, andevaporation was repeated thoroughly under vacuum. The crude product waspurified by silica gel chromatography to give the tricyclic amine as awhite solid (400 mg, 1.9 mmol, 76%).

A mixture of the tricyclic amine (400 mg, 1.9 mmol),3,6-dichlorotrimellitic anhydride (248 mg, 0.95 mmol) and polyphosphoricacid (1 g) was heated to 180° C. for 2 hr. After cooling to roomtemperature, the solid was dissolved in aqueous NaOH (1M, 7.5 ml). Theproduct was precipitated with aqueous HCl (2M, 7.5 ml). The solid wascollected by filtration and purified by reverse-phase HPLC to give thedye, Abs. max 638 nm). The regiochemistry of the 5 and 6 carboxyl groupsof the isomers were not assigned. Isomer 1 (32 mg, 5%) and Isomer 2 (65mg, 10%) were separable by reverse-phase HPLC.

Example 5

Preparation of dR139-Labeled Dideoxyadenosinetriphosphate, Formula VIII

A solution of ddATP-NH₂ (5 μL, 20 mM, 0.1 μmol) (Hobbs 1989, 1992),dR139-NHS (0.15 μmol) and 250 mM carbonate/bicarbonate buffer, pH 9 (5μL) were mixed. After 10 min at room temperature the solution wassubjected to HPLC with an anion-exchange column and eluted with agradient of 40% acetonitrile/60% 0.1 M triethylammonium bicarbonate to40% acetonitrile/60% 1.5 M triethylammonium bicarbonate to remove freedye. The fraction containing dye-labeled nucleotide and unlabelednucleotide was concentrated in a vacuum centrifuge and subjected to asecond HPLC using a reverse-phase column. The unlabeled nucleotide andeach, dye isomer of dye-labeled nucleotide were separated using anelution gradient of 15% acetonitrile/85% 0.1 M triethylammonium acetateto 35% acetonitrile/65% 0.1 M triethylammonium acetate. The solutionscontaining dye-labeled nucleotide were concentrated in a vacuumcentrifuge, redissolved in 10 mM carbonate/bicarbonate buffer, pH 9, andquantified by measuring the absorbance of the solution in a UV/Visiblespectrophotometer. Yields were approximately 1%.

Example 6

Preparation of Dye-Labeled Oligonucleotide, Formula XI

A solution of 5′-aminohexyl-functionalized oligonucleotide, (10 μL, 1mM) and dR139-NHS (10 μl, 12 mM in methylsulfoxide) andcarbonate/bicarbonate buffer (2 μL, 1 M) were combined. The aminohexylderivatized primer was prepared by automated solid-phase DNA synthesisusing Aminolink-2 in the last cycle of the synthesis (PE Biosystems).After 10 min at room temperature the solution was subjected to gelfiltration on Sephadex G-25 to separate free dye. The fractioncontaining dye-labeled oligonucleotide and unlabeled oligonucleotide wascollected and subjected to HPLC purification on a reverse-phase column.The unlabeled oligonucleotide and each dye isomer of dye-labeledoligonucleotide were separated using an elution gradient of 10%acetonitrile/85% 0.1 M triethylammonium acetate to 30% acetonitrile/65%0.1 M triethylammonium acetate. The solutions containing dye-labeledoligonucleotide were concentrated in a vacuum centrifuge, andredissolved in a buffer containing 10 mM Tris, 1 mM EDTA, pH 7.0 (TE).

Example 7

Sequencing Reactions Utilizing the 4,7-DichlororhodamineDideoxynucleotide Terminators of the Invention

Dye terminator reactions were conducted with AmpliTaq® DNA Polymerase,FS following the basic protocols in the ABI PRISM™ Dye Terminator CycleSequencing Core-Kit Manual (PE Biosystems). (The FS enzyme is arecombinant thermus aquaticus DNA polymerase-having two pointmutations—G46D and F667Y). All reagents except the dNTP mix and dyeterminators were from an ABI PRISM™ Dye Terminator Cycle Sequencing CoreKit (PE Biosystems). A premix of reaction components was prepared asfollows, where volumes are on a per reaction basis:

5X Buffer   4 μl dNTP mix   1 μl Template: pGEM ®-3Zf(+), 0.2 μg/μL   5μl Primer: −21 M13 (forward), 0.8 pmol/μL   2 μl AmpliTaq DNAPolymerase, FS 0.5 μl H₂O 2.5 μl

Reactions were set up in 0.5 ml tubes for the Perkin-Elmer 480 DNAThermal Cycler (PE Biosystems). Total reaction volumes were 20 μl,including 15 μl of the above reaction premix, an appropriate amount ofdye labeled terminator, and water. Dye terminator reactions were set upwith either 1 pmole of dye terminator for A and G terminators or with 15pmole of dye terminator for C and T terminators, with dyes of thepresent invention. In a few cases, the dye terminators for C or T wereat too low a concentration, such that 5 μL resulted in less than 15pmole of dye terminator. In these cases, 5 μl of dye terminator was usedand no water was added to the reaction. 30 μL of mineral oil was addedto the top of each reaction volume to reduce evaporation duringthermocycling. Reactions were thermocycled for 25 cycles as follows: 96°C. for 30 sec, 50° C. for 15 sec, 60° C. for 4 min; followed by a 4° C.hold cycle.

All reactions were purified by spin-column purification on Centri-Sepspin columns (Princeton Separations, Adelphia, N.J.). Gel material inthe column was hydrated with 0.8 mL of deionized water for at least 30minutes at room temperature. After the columns were hydrated, and it wasapparent that no bubbles were trapped in the gel material, the upper-endcap and then the lower-end cap were removed. The column was allowed todrain by gravity. Columns were then inserted into the wash tubesprovided in the Centi-Sep kit and centrifuged in a variable speedmicrocentrifuge (Eppendorf Model 5415) at 1300×g for 2 minutes. Columnswere removed from the wash tubes and inserted into sample collectiontubes. The reaction mixture was carefully removed from under the oilusing a glass pipette and loaded on top of the Centri-Sep column.Columns were centrifuged for 2 minutes. Samples were dried in a vacuumcentrifuge.

The dried samples were resuspended in 25 μl of Template. SuppressionReagent (PE Biosystems), vortexed, heated to 95° C. for 2 minutes,cooled on ice, vortexed again, and centrifuged (13,000×g). 10 μl of thepurified sample was aliquoted into sample vials adapted for use with thePE ABI PRISM™ 310 Genetic Analyzer (PE Biosystems). Electrophoresis onthe Model 310 used a 61 cm long, 50 μm ID uncoated fused silicacapillary having a length to the detector of 50 cm. The capillary wasfilled with a solution of a linear dimethylpolyacrylamide (DMA) sievingpolymer (Madabhushi), buffer, and containing nucleic acid denaturants(PE Biosystems). Samples were electrokinetically injected for 30 sec at2.5 kV. Electrophoresis was performed for 2 hr at 12.2 kV with thecapillary temperature maintained at 42° C.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Although only a few embodiments have been described in detail above,those having ordinary skill in the organic chemical art will clearlyunderstand that many modifications are possible in the preferredembodiment without departing from the teachings thereof. All suchmodifications are intended to be encompassed within the followingclaims.

1. A labeled nucleotide according to structural formula (VII):

wherein: B is selected from the group consisting of a 7-deazapurinenucleotide base attached to the illustrated furan at its N9 position, apurine nucleotide base attached to the furan of structural formula (VII)at its N9 position and a pyrimidine nucleotide base attached to thefuran of structural formula (VII) at its N1 position; D is a4,7-dichlororhodamine dye according to structural formula (VI):

wherein: R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁸ are each,independently of one another, selected from the group consisting ofhydrogen, fluorine, chlorine, methyl, ethyl, lower alkyl, lower alkene,lower alkyne, cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino,amido, nitrile, lower alkoxy and combinations thereof, or,alternatively, R⁷ and R⁸ or R¹³ and R¹⁴ may be taken together for forman oxo, sulfoxo, imminium or alkyliminium group; R¹¹ and R¹⁷ are each,independently of one another, selected from the group consisting ofhydrogen, lower alkyl, alkyl sulfonate, alkyl carboxylate, lower alkene,lower alkyne, cycloalkyl, phenyl, aryl and combinations thereof; and X¹,X² and X³ are each, independently of one another, selected from thegroup consisting of hydrogen, chlorine, fluorine, lower alkyl, amine,amide, carboxylate, sulfonate and hydroxymethyl, with the proviso thatone of R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, X¹, X²or X³ comprises a linkage linking D to B; W¹ and W² are each,independently of one another, selected from the group consisting ofhydrogen and OH; W³ is selected from the group consisting of OH, OPO₃,OP₂O₆, OP₃O₉, and analogs thereof.
 2. The labeled nucleotide of claim 1in which D is linked to the 8-position of B when B is a purinenucleotide base, D is linked to the 7-position of B when B is a7-deazapurine nucleotide base and D is linked to the 5-position of Bwhen B is a pyrimidine nucleotide base.
 3. The labeled nucleotide ofclaim 1 in which W³ is selected from the group consisting ofphosphorothioate, phosporoanilidate, phosphoroanilothioate andphosphoramidate.
 4. The labeled nucleotide of claim 1 in which W¹ ishydrogen, W² is OH and W³ is OP₃O₉.
 5. The labeled nucleotide of claim 1in which W¹ and W² are each hydrogen and W³ is OP₃O₉.
 6. The labelednucleotide of claim 1 in which the linkage linking B and D comprises aacetylenic amido or an alkenic amido linkage.
 7. The labeled nucleotideof claim 1 in which the linkage linking B and D is selected from thegroup consisting of —C≡C—CH₂—NH—C(O)—, —C≡C—CH₂—NH—C(O)—(CH₂)₅—NH—C(O)—,—CH═CH—C(O)—NH—(CH₂)₅—NH—C(O)—, —C≡C—CH₂—O—CH₂CH₂—NH—C(O)—,—C≡C—CH₂—O—CH₂CH₂—O—CH₂CH₂—NH—C(O)— and —C≡C—Ph—O—CH₂CH₂—NH—C(O)—, wherePh is 1,4-phenylene.
 8. The labeled nucleotide of any one of claims 1-7in which X¹ is carboxylate; one of X² or X³ is hydrogen and the otherone of X² or X³ comprises the linkage linking D to B.
 9. The labelednucleotide of any one of claims 1-7 in which R⁷, R⁸, R⁹, R¹⁰, R¹³, R¹⁴,R¹⁵ and R¹⁶ are each, independently of one another, selected from thegroup consisting of hydrogen, methyl and ethyl.
 10. The labelednucleotide of any one of claims 1-7 in which R¹¹ and R¹² are each,independently of one another, selected from the group consisting ofmethyl and phenyl.
 11. The labeled nucleotide of any one of claims 1-7in which R⁷, R⁸, R⁹, R¹⁰, R¹³, R¹⁴, R¹⁵ and R¹⁶ are each, independentlyof one another, selected from hydrogen, methyl and ethyl.
 12. Thelabeled nucleotide of any one of claims 1-7 in which R⁷, R⁸, R¹⁰, R¹³,R¹⁴ and R¹⁷ are each, independently of one another, selected from thegroup consisting of hydrogen and methyl; R⁹ and R¹⁵ are each hydrogen;R¹¹ and R¹⁶ are each methyl or phenyl; and R¹² and R¹⁸ are eachhydrogen.
 13. A labeled polynucleotide containing a nucleotide accordingto structural formula (XI):

wherein: B is selected from the group consisting of a 7-deazapurinenucleotide base attached to the illustrated furan at its N9 position, apurine nucleotide base attached to the furan of structural formula (XI)at its N9 position and a pyrimidine nucleotide base attached to thefuran of structural formula (XI) at its N1 position; D is a4,7-dichlororhodamine dye according to structural formula (VI):

wherein: R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁸ are each,independently of one another, selected from the group consisting ofhydrogen, fluorine, chlorine, methyl, ethyl, lower alkyl, lower alkene,lower alkyne, cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino,amido, nitrile, lower alkoxy and combinations thereof, or,alternatively, R⁷ and R⁸ or R¹³ or R¹⁴ may be taken together for form anoxo, sulfoxo, imminium or alkyliminium group; R¹¹ and R¹⁷ are each,independently of one another, selected from the group consisting ofhydrogen, lower alkyl, alkyl sulfonate, alkyl carboxylate, lower alkene,lower alkyne, cycloalkyl, phenyl, aryl and combinations thereof; and X¹,X² and X³ are each, independently of one another, selected from thegroup consisting of hydrogen, chlorine, fluorine, lower alkyl, amine,amide, carboxylate, sulfonate and hydroxymethyl, with the proviso thatone of R⁷, R⁸, R⁹, R10, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, X¹, X²or X³ comprises a linkage linking D to B Z¹ is selected from the groupconsisting of hydrogen, OH and OCH₃; Z² is selected from the groupconsisting of hydrogen, OH, OPO₃, OP₂O₆, OP₃O₉ and Nuc¹, where Nuc¹ is anucleosoide, nucleotide or polynucleotide which is linked to structuralformula (XI) by a phosphodiester linkage or an analog thereof, thelinkage being attached to the 5′-position of Nuc¹; and Z³ is selectedfrom the group consisting of hydrogen, PO₃, phosphate analogs and Nuc²,where Nuc² is a nucleoside, nucleotide or polynucleotide which is linkedto structural formula (XI) by a phosphodiester linkage or an analogthereof, the linkage being attached to the 3′-position of Nuc².
 14. Thelabeled polynucleotide of claim 13 in which D is linked to the8-position of B when B is a punne nucleotide base, D is linked to the7-position of B when B is a 7-deazapurine nucleotide base and D islinked to the 5-position of B when B is a pyrimidine nucleotide base.15. The labeled polynucleotide of claim 13 in which the illustratednucleotide is a 5′-terminal nucleotide.
 16. The labeled polynucleotideof claim 13 in which the illustrated nucleotide is a 3′-terminalnucleotide.
 17. The labeled polynucleotide of claim 13 in which theillustrated nucleotide is an internal nucleotide of the polynucleotide.18. The labeled polynucleotide of claim 13 in which the linkage linkingB and D comprises a acetylenic amido or an alkenic amido linkage. 19.The labeled polynucleotide of claim 13 in which the linkage linking Band D is selected from the group consisting of —C≡C—CH₂—NH—C(O)—,—C≡C—CH₂—NH—C(O)—(CH₂)₅—NH—C(O)—, —CH═CH—C(O)—NH—(CH₂)₅—NH—C(O)—,—C≡C—CH₂—O—CH₂CH₂—NH—C(O)—, —C≡C—CH₂—O—CH₂CH₂—O—CH₂CH₂—NH—C(O)— and—C≡C—Ph—O—CH₂CH₂—NH—C(O)—, where Ph is 1,4-phenylene.
 20. The labelednucleotide of any one of claims 13-19 in which X¹ is carboxylate; one ofX² or X³ is hydrogen and the other one of X² or X³ comprises the linkagelinking D to B.
 21. The labeled polynucleotide of any one of claims13-19 in which R⁷, R⁸, R⁹, R¹⁰, R¹³, R¹⁴, R¹⁵ and R¹⁶ are each,independently of one another, selected from the group consisting ofhydrogen, methyl and ethyl.
 22. The labeled nucleotide of any one ofclaims 13-19 in which R¹¹ and R¹² are each, independently of oneanother, selected from the group consisting of methyl and phenyl. 23.The labeled nucleotide of any one of claims 13-19 in which R⁷, R⁸, R⁹,R¹⁰, R¹³, R¹⁴, R¹⁵ and R¹⁶ are each, independently of one another,selected from hydrogen, methyl and ethyl.
 24. The labeled nucleotide ofany one of claims 13-19 in which R⁷, R⁸, R¹⁰, R¹³, R¹⁴ and R¹⁷ are each,independently of one another, selected from the group consisting ofhydrogen and methyl; R⁹ and R¹⁵ are each hydrogen; R¹¹ and R¹⁶ are eachmethyl or phenyl; and R¹² and R¹⁸ are each hydrogen.
 25. A labelednucleotide according to structural formula (VII):

wherein: B is selected from the group consisting of a 7-deazapurinenucleotide base attached to the illustrated furan at its N9 position, apurine nucleotide base attached to the furan of structural formula (VII)at its N9 position and a pyrimidine nucleotide base attached to thefuran of structural formula (VII) at its N1 position; D is a4,7-dichlororhodamine dye according to structural formula (I):

wherein: R¹, R², R³, R⁴, R⁵ and R⁶, when taken alone, are each,independently of one another, selected from the group consisting ofhydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne,cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino, amido, nitrile,lower alkoxy and combinations thereof, or, alternatively, R¹ and R²and/or R⁴ and R⁵ are taken together to form a benzo group; Y¹, Y², Y³and Y⁴, when taken alone, are each, independently of one another,selected from the group consisting of hydrogen, lower alkyl, alkylsulfonate, alkyl carboxylate and cycloalkyl, or, alternatively, Y¹ andR², Y² and R¹, Y³ and R³ and/or Y⁴ and R⁴, are taken together and areeach, independently of one another selected from the group consisting ofethano, propano and substituted forms thereof; X¹, X² and X³ are each,independently of one another, selected from the group consisting ofhydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonate andhydroxymethyl, with the proviso that one of R¹, R², R³, R₄, R⁵, R₆, X¹,X² or X³ comprises a linkage linking D to B; W¹ and W² are each,independently of one another, selected from the group consisting ofhydrogen and OH; W³ is selected from the group consisting of OH, OPO₃,OP₂O₆, OP₃O₉, and analogs thereof.
 26. The labeled nucleotide of claim25 in which D is linked to the 8-position of B when B is a purinenucleotide base, D is linked to the 7-position of B when B is a7-deazapurine nucleotide base and D is linked to the 5-position of Bwhen B is a pyrimidine nucleotide base.
 27. The labeled nucleotide ofclaim 25 in which W³ is selected from the group consisting ofphosphorothioate, phosporoanilidate, phosphoroanilothioate andphosphoramidate.
 28. The labeled nucleotide of claim 25 in which W¹ ishydrogen, W² is OH and W³ is OP₃O₉.
 29. The labeled nucleotide of claim25 in which W¹ and W² are each hydrogen and W³ is OP₃O₉.
 30. The labelednucleotide of claim 25 in which the linkage linking B and D comprises aacetylenic amido or an alkenic amido linkage.
 31. The labeled nucleotideof claim 25 in which the linkage linking B and D is selected from thegroup consisting of —C≡C—CH₂—NH—C(O)—, —C≡C—CH₂—NH—C(O)—(CH₂)₅—NH—C(O)—,—CH═CH—C(O)—NH—(CH₂)₅—NH—C(O)—, —C≡C—CH₂—O—CH₂CH₂—NH—C(O)—,—C≡C—CH₂—O—CH₂CH₂—O—CH₂CH₂—NH—C(O)— and —C≡C—Ph—O—CH₂CH₂—NH—C(O)—, wherePh is 1,4-phenylene.
 32. The labeled nucleotide of any one of claims25-31 in which X¹ is carboxylate; one of X² or X³ is hydrogen and theother one of X² or X³ comprises the linkage linking D to B.
 33. Thelabeled nucleotide of any one of claims 25-31 in which R², R³, R⁵, R⁶,Y¹ and Y³ are each hydrogen; Y² and R¹ are taken together to form apropano group; Y⁴ and R⁴ are taken together to form a propano group; X¹is carboxylate; and one of X² and X³ is hydrogen and the other comprisesthe linkage to B.
 34. The labeled nucleotide of any one of claims 25-31in which R², R³, R⁵ and R⁶ are each hydrogen; Y¹ and Y³ are each methyl;Y² and R¹ are taken together to form an propane group; Y⁴ and R⁴ aretaken together to from a propane group; X¹ is carboxylate; and one of X²and X³ is hydrogen and the other comprises the linkage to B.
 35. Thelabeled nucleotide of any one of claims 25-31 in which R¹, R⁴, R⁵ and R⁶are each hydrogen; Y² and Y⁴ are each ethyl; Y¹ and R² are takentogether to form an ethano group; Y³ and R³ are taken together to forman ethano group; X¹ is carboxylate; and one of X² and X³ is hydrogen andthe other comprises the linkage to B.
 36. The labeled nucleotide of anyone of claims 25-31 in which R¹, R⁴, R⁵ and R⁶ are each hydrogen; Y² andY⁴ are each —(CH₂)₅—COOH; Y¹ and R² are taken together to form a—CH₂—C(CH₃)₂— group; Y³ and R³ are taken together to form a—CH₂—C(CH₃)₂— group; X¹ is carboxylate; and one of X² and X³ is hydrogenand the other comprises the linkage to B.
 37. The labeled nucleotide ofany one of claims 25-31 in which R¹, R⁴, R⁵ and R⁶ are each hydrogen; Y²and Y⁴ are each —CH₂-Ph—COOH, where Ph is 1,4-phenylene; Y¹ and R² aretaken together to form a —CH₂—C(CH₃)₂— group; Y³ and R³ are takentogether to form a —CH₂—C(CH₃)₂— group; X¹ is carboxylate; and one of X²and X³ is hydrogen and the other comprises the linkage to B.
 38. Thelabeled nucleotide of any one of claims 25-31 in which R¹, R², R³, R⁴,R⁵ and R⁶ are each hydrogen; Y¹ and Y² are taken together to form abutano group; Y³ and Y⁴ are taken together to form abutano group; X¹ iscarboxylate; and one of X² and X³ is hydrogen and the other comprisesthe linkage to B.
 39. A labeled polynucleotide containing a nucleotideaccording to structural formula (XI):

wherein: B is selected from the group consisting of a 7-deazapurinenucleotide base attached to the furan of structural formula (XI) at itsN9 position, a purine nucleotide base attached to the furan ofstructural formula (XI) at its N9 position, and a pyrimidine nucleotidebase attached to the furan of structural formula (XI) at its N1position; D is a 4,7-dichlororhodamine dye according to structurallformula (I):

wherein: R¹, R₂, R³, R⁴, R⁵ and R⁶, when taken alone, are each,independently of one another, selected from the group consisting ofhydrcgen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne,cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino, amido, nitrile,lower alkoxy and combinations thereof, or, alternatively, R¹ and R⁶and/or R⁴ and R⁵ are taken together to form a benzo group; Y¹, Y², Y³and Y⁴, when taken alone, are each, independently of one another,selected from the group consisting of hydrogen, lower alkyl, alkylsulfonate, alkyl carboxylate and cycloalkyl, or, alternatively, Y¹ andR₂, Y² and R¹, Y³ and R³ and/or Y⁴ and R⁴, are taken together and areeach, independently of one another selected from the group consisting ofethano, propano and substituted forms thereof X¹, X² and X³ are each,independently of one another, selected from the group consisting ofhydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonate andhydroxymethyl, with the proviso that one of R¹, R², R³, R⁴, R⁵, R⁶, X¹,X² or X³ comprises a linkage linking D to B; Z₁ is selected from thegroup consisting of hydrogen and OH; Z₂ is selected from the groupconsisting of hydrogen, OH, OPO₃, OP₂O₆, OP₃O₉ and Nuc¹, where Nuc¹ is anucleoside, nucleotide or polynucleotide which is linked to structuralformala (XI) by a phosphodiester linkage or an analog thereof; thelinkage being attached to the 5′-position of Nuc¹; and Z³ is selectedfrom the group consisting of hydrogen, PO₃, phosphate analogs and Nuc²,where Nuc² is a nucleoside, nucleotide or polynucleotide which is linkedto structural formal (XI) by a phosphodiester linkage or an analogthereof, the linkage being attached to the 3′-position of Nuc².
 40. Thelabeled polynucleotide of claim 39 in which D is linked to the8-position of B when B is a purine nucleotide base, D is linked to the7-position of B when B is a 7-deazapurine nucleotide base and D islinked to the 5-position of B when B is a pyrimdine nucleotide base. 41.The labeled polynucleotide of claim 39 in which the illustratednucleotide is a 5′-terminal nucleotide.
 42. The labeled polynucleotideof claim 39 in which the illustrated nucleotide is a 3′-terminalnucleotide.
 43. The labeled polynucleotide of claim 39 in which theillustrated nucleotide is an internal nucleotide of the polynucleotide.44. The labeled polynucleotide of claim 37 in which the linkage linkingB and D comprises a acetylenic amido or an alkenic amido linkage. 45.The labeled polynucleotide of claim 39 in which the linkage linking Band D is selected from the group consisting of —C≡C—CH₂—NH—C(O)—,—C≡C—CH₂—NH—C(O)—(CH₂)₅—NH—C(O)—, —CH═CH—C(O)—NH—(CH₂)₅—NH—C(O)—,—C≡C—CH₂—O—CH₂CH₂—NH—C(O)—, —C≡C—CH₂—O—CH₂CH₂—O—CH₂CH₂—NH—C(O)— and—C≡C—Ph—O—CH₂CH₂—NH—C(O)—, where Ph is 1,4-phenylene.
 46. The labelednucleotide of any one of claims 39-45 in which X¹ is carboxylate; one ofX² or X³ is hydrogen and the other one of X² or X³ comprises the linkagelinking D to B.
 47. The labeled nucleotide of any one of claims 39-45 inwhich R², R³, R⁵, R⁶, Y¹ and Y³ are each hydrogen; Y² and R¹ are takentogether to form a propano group; Y⁴ and R⁴ are taken together to form apropano group; X¹ is carboxylate; and one of X² and X³ is hydrogen andthe other comprises the linkage to B.
 48. The labeled nucleotide of anyone of claims 39-45 in which R², R³, R⁵ and R⁶ are each hydrogen; Y¹ andY³ are each methyl; Y² and R¹ are taken together to form an propanegroup; Y⁴ and R⁴ are taken together to from a propane group; X¹ iscarboxylate; and one of X² and X³ is hydrogen and the other comprisesthe linkage to B.
 49. The labeled nucleotide of any one of claims 39-45in which R¹, R⁴, R⁵ and R⁶ are each hydrogen; Y² and Y⁴ are each ethyl;Y¹ and R² are taken together to form an ethano group; Y³ and R³ aretaken together to form an ethano group; X¹ is carboxylate; and one of X²and X³ is hydrogen and the other comprises the linkage to B.
 50. Thelabeled nucleotide of any one of claims 39-45 in which R¹, R⁴, R⁵ and R⁶are each hydrogen; Y² and Y⁴ are each —(CH₂)₅—COOH; Y¹ and R² are takentogether to form a —CH₂—C(CH₃)₂— group; Y³ and R³ are taken together toform a —CH₂—C(CH₃)₂— group; X¹ is carboxylate; and one of X² and X³ ishydrogen and the other comprises the linkage to B.
 51. The labelednucleotide of any one of claims 39-45 in which R¹, R⁴, R⁵ and R⁶ areeach hydrogen; Y² and Y⁴ are each —CH₂—Ph—COOH, where Ph is1,4-phenylene; Y¹ and R² are taken together to form a —CH₂—C(CH₃)₂—group; Y³ and R³ are taken together to form a —CH₂—C(CH₃)₂— group; X¹ iscarboxylate; and one of X² and X³ is hydrogen and the other comprisesthe linkage to B.
 52. The labeled nucleotide of any one of claims 39-45in which R¹, R², R³, R⁴, R⁵ and R⁶ are each hydrogen; Y¹ and Y² aretaken together to form a butano group; Y³ and Y⁴ are taken together toform a butano group; X¹ is carboxylate; and one of X² and X³ is hydrogenand the other comprises the linkage to B.